This is a proposal from a group of five NIH funded principal investigators requesting funds to purchase a commercially available inverted two-photon laser scanning microscope (TPLSM) system that will be primarily utilized for BSL2 projects. This shared instrument will provide new capabilities for deep tissue imaging in visceral organs (liver, spleen and intestines) in mice infected with BSL2 level pathogens. These new capabilities are essential for specific projects focusing on host-defense against bacterial, viral and parasitic attack. We propose to characterize the immune response to Listeria in live mouse spleen, lymph nodes and liver (Project 1), the immune response to Mycobacteria in the lymph nodes and lung tissue (Project 2), the immune response to human malarial parasites in reconstituted human tissue (Project 3), determine the migration patterns of inflammatory T cell populations in the gut associated lymphoid tissues during infection with bacterial and protozoan pathogens (Project 4), and determine the impact of Staphylococcal toxins on innate immune cells during abscess formation (Project 5). For each of the projects, capabilities unique to the new system will be invaluable. The key advantages of the new system compared to existing systems are 1) inverted configuration, which is ideal for intravital microscopy of the visceral organs, and 2) it will be housed in a BSL-2 facility. The instrument will be housed on the second floor of the Skirball institute where 3 of the major users and the facility manager are located. The facility manager has experience with operating BSL3 microscopy equipment and has over 30 years experience with laser based instrumentation. This device will have long-term institutional support to cover the service contracts. Time will also me made available to other NIH funded researchers at NYU and in the NY area who need the unique capabilities of the Leica SP5 TPLSM system. The inverted system is unusual for intravital microscopy systems, but extensive justification is provided for why the inverted system is needed for the imaging of tissues that are major targets for pathogen entry and colonization. The system will provide vital support for novel studies on host-defense and host- pathogen interactions. Hypotheses that require dynamic microscopic information about pathogen, host cell and tissue changes during infection can be addressed. The impact on public health will be to develop better strategies to fight infections and reduce the incidence of pathogen triggered autoimmunity. [unreadable] [unreadable] [unreadable]